Early studies showed that arylsulfatase A (ASA) electrophoresed in native polyacrylamide gels and stained for enzymatic activity exhibited a variety of electrophoretic patterns, some of which were more likely to be found in alcoholic patients than in non-alcoholic psychiatric and normal control subjects Hulyalkar et al., Alcoh.: Clin. Exp. Res. 8:337-341 (1984)!. However, lacking any biochemical explanation for these observations, no correlation with a genetic basis or marker for alcoholism was possible.
A more severe neuropathological disease associated with a deficiency of ASA is metachromatic leukodystrophy (MLD). MLD is a debilitating genetic disease characterized by neuropsychological deficits. In late-onset MLD, the initial symptoms include attentional difficulties, hyperactivity, impulsivity, poor judgement, and emotional lability Shapiro et al., Neurology 44:662-665 (1994)!. MLD patients display a characteristic demyelination pathology resulting from increased levels of sulfatides. This glycolipid comprises a significant proportion of the myelin sheath and accumulates in various tissues of individuals with MLD. MLD is caused by the lack of the enzyme activity of arylsulfatase A (ASA, EC 3.1.6.8), a lysosomal glycoprotein which catalyzes the desulfation of sulfatides, the first step in sulfatide catabolism.
Some individuals exhibit much reduced levels of ASA activity, but appear to catabolize sulfatides adequately and lack apparent MLD-related neurological symptoms Kolodny, E. H., The Metabolic Basis of Inherited Disease, eds., pp. 1721-1750 (1989)!. These people are pseudodeficient for ASA (PD-ASA) and possess an ASA gene which has two A-G transitions. One of these mutations results in an Asn.sub.350 to Ser conversion, causing a loss of a potential N-glycosylation site, and the other in a polyadenylation signal consensus sequence alteration, resulting in a reduction of a 2.1 Kb message Gieselmann et al., Proc. Natl. Acad. Sci. USA 86:9436-9440 (1989)!. The PD-ASA gene frequency, at approximately 10%, is quite common Nelson et al., Hum. Genet. 87:87-88 (1991)!. A number of multiband electrophoretic variants of ASA are found in the general population Poretz, et al., Biochem. J. 287:979-983 (1992)!. Previous work demonstrated that the electrophoretic (non-denaturing) pattern is due to a heterogeneous mixture of ASA isoforms with variable degrees of phosphorylation of the N-glycan moieties, and hypothesized that the variant forms may reflect differences in both the structure of the carbohydrate units and polypeptide of the enzyme (Poretz et al., supra; Park et al., Alcohol. Clin. Exper. Res. 20:234-239 (1996)!.
The pseudodeficiency mutations do not cause MLD but reduce the enzyme activity of ASA sufficiently to complicate the diagnosis of MLD and MLD carrier status in families where they occur Wenger & Louie, Dev. Neurosci., 13:216-221 (1991)!. While the polyadenylation signal sequence mutation has been proposed to be the cause of the reduced ASA activity in pseudodeficiency Gieselmann et al., supra (1989)!, there is evidence that the N-glycosylation site mutation reduces ASA enzyme activity to a greater extent Shen et al., Am. J. Med. Genet., 45:631-637 (1993)1. This is significant as the N-glycosylation site mutation can occur in the absence of the polyadenylation signal site mutation (Nelson et al., supra; Shen et al., supra!. The ability to distinguish this mutation is therefore important in diagnosis and risk determination in families with MLD Shen et al., supra; Park et al., supra (1996)!.
Previously, allele specific tests have been described Gieselmann et al., supra (1989); Gieselmann et al., Hum. Genet. 86:251 (1991)!. However, no clear relationship between neurologic disease and enzyme deficiency has been conclusively established Kolodny, in Metabolic Basis of Inherited Disease, Vol. II, C. R. Scriber et al. (Eds.), McGraw-Hill: New York. pp. 1721-1740 (1989)!. In particular, homozygosity for the PD-ASA allele has been reported to bear no clinical consequence, and that homozygosity for the PD-ASA allele is no more frequent among neuropsychiatric patients than normal controls Hohenschutz et al., Am. J. Med. Genet. 31:169-175 (1988)!. Individuals who present with an ASA.sup.negative /PD-ASA genotype (i.e., heterozygous MLD) may have a greater incidence of clinical abnormalities Hohenschutz et al., Hum. Genet. 82:4548 (1989)!. A more recent publication disputes this hypothesis Penzien et al., Am. J. Hum. Genet. 52:557-564 (1993)!.
An important breakthrough in the prevention of alcoholism came with the recognition that homozygosity for the PD-ASA allele is associated with a predisposition to alcoholism (International Patent Publication WO 96/07098, published Mar. 7, 1996, by Manowitz et al.; U.S. patent application Ser. No. 08/299,187 filed Aug. 31, 1994; provisional U.S. Patent Application No. 60/001,300, file Jun. 21, 1995; and U.S. application Ser. No. 08/666,029, filed Jun. 19, 1996; each of which is incorporated herein in its entirety). This discovery represented the first identification of a genetic association of ASA with alcoholism. Previously, biochemical differences in ASA (i.e., the extent of glycosylation) were attributed to the effects of alcohol on protein processing and glycosylation. In particular, homozygosity for a mutation in a residue 350 N-glycosylation site of ASA, or both this mutation and a mutation of the polyadenylation signal sequence, correlates with greater susceptibility to alcoholism. The data presented in that publication demonstrate that humans who are homozygous for a genetic mutation of ASA that results in absence of an N-linked glycan at amino acid residue 350 of ASA are approximately 8 to approximately 18 times more likely to be found with alcoholics than non-alcoholic individuals.